Container for germ layer formation and method of forming germ layer

ABSTRACT

The invention relates to a vessel for embryoid formation used for forming embryoid bodies from ES cells easily without complicated technique, and to a method for forming embryoid bodies easily and efficiently using the vessel. The method includes the steps of (A) providing a vessel for embryoid formation having a coating layer formed from a compound having a particular PC-like group on a vessel surface defining a region for floating culture of ES cells, and (B) floating culturing ES cells in the vessel to form embryoid bodies.

FIELD OF ART

The present invention relates to a vessel for embryoid formation for usein forming embryoid bodies, and a method for forming embryoid bodies.

BACKGROUND ART

Embryonic stem cells (ES cells) are capable of differentiating intovarious types of cells even in vitro. In vitro differentiation of EScells is performed by floating culture to form pseudo-embryos, calledembryoid bodies, or by coculture with cells, such as stromal cells, thatsupport differentiation and proliferation of ES cells. It is known thatES cells differentiate into various types of cells when the cells arecultured to high density without LIF (Leukemia Inhibitory Factor), andthen floating cultured so as not to adhere to a culture vessel, such asa petri dish, to form cell aggregates. The cell aggregates formed byfloating culture are called embryoid bodies (EB), and the floatingculture is the most common method for differentiating ES cells in vitro.

An embryoid body has a ball-like structure composed of a bilayer ofcells. The outer layer corresponds to visceral endoderm, the inner layercorresponds to embryonic ectoderm, and the two endoderms are separatedby a basement membrane. This structure is quite similar to that of acylindrical embryo, which is a day 6 mouse embryo. As far as thissimilarity is concerned, this structure resembles the normal stage ofembryogenesis. In embryoid bodies, mesoderm is also induced, andcardiomyocytes, blood cells, and even primitive vascular networks aredeveloped. When plated on a culture petri dish and cultured further, theembryoid bodies differentiate into various types of cells, includingneurons, keratinocytes, chondrocytes, adipocites, and the like. It hasrecently been confirmed that the cells that differentiate via formationof embryoid bodies are differentiated not only into somatic cells, butalso into a germ cell lineage. As such, formation of embryoid bodies isuseful for demonstrating pluripotency of ES cells.

For embryoid formation, so-called a “hanging drop method” is widelyused, which is devised to prevent adhesion of ES cells to a culturevessel. There are known hanging drop method 1, wherein ES cells areadded to and cultured in the drops hanging from the lid of a glasscontainer, and hanging drop method 2, wherein ES cells are placed overmineral oil previously placed in a culture vessel, and cultured. Inhanging drop method 1; however, the hanging drops must be prevented fromfalling, or the interface between the mineral oil and the overlaid cellsuspension must be prevented from being disrupted, which causes extremecomplexity in culture preparation and handling. In hanging drop method 2using mineral oil, on the other hand, no microscopic examination isallowed before the generated embryoid bodies are transferred to anotherculture vessel, which impedes researches in embryogenesis.

Phosphorylcholine group-containing polymers have been revealed to haveproperties ascribable to their phospholipid-like structure originatedfrom biomembranes, such as blood compatibility, complement activation,and nonadsorbability of biomaterials, and development of bio-relatedmaterials making good use of such functions has been actively made. Forexample, Patent Publication 1 discloses a method of producing2-methacryloyloxyethyl phosphorylcholine (abbreviated as MP/Chereinbelow) and excellent biocompatibility of polymers thereof. PatentPublication 2 discloses usefulness of copolymers of MP/C andmethacrylate as medical materials due to their ability to hardly allowplatelet adhesion or aggregation and plasma protein adhesion. PatentPublication 3 discloses medical materials prepared from a copolymerhaving a phosphorylcholine-like group in its side chain. PatentPublications 4 and 5 disclose excellent biocompatibility achieved bycoating a resin surface with a polymer having a phosphorylcholine-likegroup. Patent Publication 6 discloses a separating agent and a method ofseparation and collection for separating and collecting blood cells,cell lines, or primary culture cells, using polyethylene terephthalatecoated with a polymer having a phosphorylcholine-like group.

It is not known, however, to use a vessel coated with a polymer having aphosphorylcholine-like group, for floating culture of ES cells.

Patent Publication 1: JP-54-36025-A

Patent Publication 2: JP-3-39309-A

Patent Publication 3: JP-9-183819-A

Patent Publication 4: JP-6-502200-A

Patent Publication 5: JP-7-502053-A

Patent Publication 6: JP-2002-098676-A

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a vessel forembryoid formation that is used for easy formation of embryoid bodiesfrom ES cells without complicated techniques.

It is another object of the present invention to provide a method forforming embryoid bodies that enables easy culture of ES cells to formembryoid bodies without complicated techniques.

According to the present invention, there is provided a vessel forembryoid formation for use in floating culture of embryonic stem cells(ES cells) to form embryoid bodies, comprising a coating layer formedfrom a compound having a phosphorylcholine-like group represented by theformula (1) (abbreviated as PC-like group hereinbelow), on a vesselsurface defining a region for floating culture of ES cells:

wherein R¹, R², and R³ are the same or different groups, and each standsfor a hydrogen atom, an alkyl or hydroxyalkyl group having 1 to 6 carbonatoms; and n is an integer of 1 to 4.

According to the present invention, there is provided a method forforming embryoid bodies, comprising the steps of:

(A) providing a vessel for embryoid formation having a coating layerformed from a compound having a PC-like group represented by the formula(1), on a vessel surface defining a region for floating culture of EScells, and

(B) floating culturing ES cells in said vessel for embryoid formation toform embryoid bodies.

According to the present invention, there is also provided use of avessel for embryoid formation for floating culture of ES cells to formembryoid bodies, said vessel comprising a coating layer formed from acompound having a PC-like group represented by the formula (1), on avessel surface defining a region for floating culture of ES cells.

In the method for forming embryoid bodies of the present invention,since the vessel for embryoid formation of the present invention is usedin culture, embryoid bodies may be formed from ES cells easily andefficiently without complicated techniques which are required forculturing ES cells by the conventional hanging drop method. Since thevessel for embryoid formation of the present invention has a coatinglayer formed from a compound having a PC-like group on a desiredsurface, the present vessel is useful for forming embryoid bodies fromES cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photocopy of a phase contrast micrograph of an embryoid bodyprepared in Example 2-1.

FIG. 2 is a photocopy of a phase contrast micrograph of an embryoid bodycultured in Comparative Example 2-1 using an untreated plate.

FIG. 3 is a photocopy of a phase contrast micrograph of an embryoid bodyformed by the hanging drop method in Comparative Example 2-2.

PREFERRED EMBODIMENTS OF THE INVENTION

The present invention will now be explained in detail.

The vessel for embryoid formation according to the present invention isfor use in floating culture of ES cells to form embryoid bodies. Thepresent vessel is characterized by its coating layer formed from acompound having a PC-like group represented by the formula (1), on avessel surface defining a region for floating culture of ES cells.

In the formula (1), R¹, R², and R³ are the same or different groups, andeach stands for a hydrogen atom, an alkyl or hydroxyalkyl group having 1to 6 carbon atoms.

The alkyl group having 1 to 6 carbon atoms may be, for example, amethyl, ethyl, propyl, butyl, pentyl, hexyl, cyclohexyl, or phenylgroup. The hydroxyalkyl group having 1 to 6 carbon atoms may be, forexample, a hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl,4-hydroxybutyl, 5-hydroxypentyl, or 6-hydroxyhexyl group.

The coating layer may be formed on a vessel surface using a compoundhaving the PC-like group represented by the formula (1) by a method of,for example, fixing a reaction reagent containing the compound having aPC-like group on a desired surface of a vessel by chemical modification,fixing a polymer having a PC-like group on a desired surface of a vesselby coating, or fixing a polymer having a PC-like group on a desiredsurface of a vessel by chemical bonding. Among these, the coating methodis particularly preferred for easy and convenient formation of a uniformcoating layer of the compound having a PC-like group.

The polymer having a PC-like group may be any polymer as long as it hasa PC-like group represented by the formula (1), and may preferably be,for example, at least one of a homopolymer of monomer (M) represented bythe formula (2) having a PC-like group, and a copolymer of monomer (M)and another monomer:

wherein R¹, R², R³, and n are the same as those in the formula (1); R⁴stands for an alkyl group having 1 to 6 carbon atoms; and R⁵ stands fora hydrogen atom or a methyl group.

Monomer (M) represented by the formula (2) may be, for example,2-((meth)acryloyloxy)ethyl-2′-(trimethylammonio) ethylphosphate,3-((meth) acryloyloxy) propyl-2′-(trimethylammonio)ethylphosphate,4-((meth)acryloyloxy)butyl-2′-(trimethylammonio) ethylphosphate,5-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethylphosphate,6-((meth)acryloyloxy) hexyl-2′-(trimethylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(triethylammonio) ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tripropylammonio) ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tributylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tricyclohexylammonio) ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(triphenylammonio) ethylphosphate,2-((meth) acryloyloxy) propyl-2′-(trimethylammonio)ethylphosphate,2-((meth)acryloyloxy)butyl-2′-(trimethylammonio) ethylphosphate,2-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethylphosphate, or2-((meth)acryloyloxy)hexyl-2′-(trimethylammonio) ethylphosphate.

Among these,2-((meth)acryloyloxy)ethyl-2′-(trimethylammonio)ethylphosphate ispreferred, and in particular,2-(methacryloyloxy)ethyl-2′-(trimethylammonio)ethylphosphate (alsocalled 2-methacryloyloxyethyl phosphorylcholine, abbreviated as MPChereinbelow) is more preferred for its availability and capability ofpreventing adhesion of ES cells to the culture vessel to facilitateexpression of their ability to form embryoid bodies.

Examples of another monomer used in preparing the copolymer may includehydrophobic monomers; (meth)acrylates containing a hydroxyl group, suchas 2-hydroxyethyl(meth)acrylate, 2-hydroxybutyl (meth)acrylate, and4-hydroxybutyl(meth)acrylate; monomers containing an ionic group, suchas acrylic acid, methacrylic acid, styrenesulfonic acid,(meth)acryloyloxyphosphonic acid, and2-hydroxy-3-(meth)acryloyloxypropyl trimethyl ammonium chloride;monomers containing nitrogen, such as (meth)acrylamide,aminoethylmethacrylate, and dimethylaminoethyl(meth)acrylate;polyethylene glycol (meth)acrylate; glycidyl(meth)acrylate; or a mixtureof two or more of these.

Examples of the hydrophobic monomers may include straight or branchedalkyl(meth)acrylate, such as methyl(meth)acrylate, ethyl(meth)acrylate,butyl(meth)acrylate, 2-ethylhexyl(meth)acrylate, lauryl(meth)acrylate,or stearyl(meth)acrylate; cyclic alkyl(meth)acrylate, such ascyclohexyl(meth)acrylate; aromatic (meth)acrylate, such asbenzyl(meth)acrylate, or phenoxyethyl(meth)acrylate; hydrophobicpolyalkylene glycol(meth)acrylate, such as polypropyleneglycol(meth)acrylate; styrene monomers, such as styrene, methylstyrene,or chloromethylstyrene; vinyl ether monomers, such as methyl vinyl etheror butyl vinyl ether; vinyl ester monomers, such as vinyl acetate orvinyl propionate; or a mixture of two or more of these.

In the copolymer, the content of the unit derived from the hydrophobicmonomers is preferably not more than 90 mol %, more preferably 20 to 90mol % of all the units of the copolymer. Copolymers having a unitderived from a hydrophobic monomer have improved elution resistance.However, if the content of the unit derived from a hydrophobic monomerexceeds 90 mol %, the amount of the PC-like group represented by theformula (1) coated on the vessel surface is too small, and sufficienteffect of the coating may not be exhibited.

The copolymer is given improved elution resistance when the copolymercontains a unit derived from monomers other than the hydrophobicmonomers. This allows use of surfactants or organic solvents in themedium or the like, which is advantageous.

For example, a copolymer prepared using glycidyl (meth)acrylate may bereacted with the amino, carboxyl, or the like groups on the vesselsurface to chemically bond the copolymer to the desired surface.

In the copolymer, the content of the units derived from monomers otherthan the hydrophobic monomers is preferably not more than 70 mol %.

The molecular weight of the homopolymer of monomer (M) represented bythe formula (2) having a PC-like group, or of the copolymer of monomer(M) and another monomer, is usually 5000 to 5000000 in weight averagemolecular weight. For effectively preventing adhesion of ES cells to theculture vessel to allow expression of their ability to form embryoidbodies, and improving the elution resistance of the polymer, themolecular weight of the polymer is preferably 100000 to 2000000.

The amount of coating in the coating layer of the present invention maybe evaluated by surface analysis. More specifically, the amount ofcoating may be evaluated by the ratio of the peak area P of phosphorusto the peak area C of carbon, i.e., the P/C value, based on the spectrummeasured by the X-ray photoelectron spectroscopy. For allowingexpression of the ability to form embryoid bodies, the P/C value ispreferably in the range of 0.002 to 0.3, more preferably 0.01 to 0.2.

The type of the vessel for embryoid formation of the present inventionis not particularly limited, and may be a conventional cell culturevessel, such as a cell culture dish, a cell culture multidish, a cellculture plate, a cell culture bag, or a cell culture flask. Forobtaining embryoid bodies of an appropriate size, a cell culture dish ora cell culture plate is particularly preferred. The material of thevessel for embryoid formation is not particularly limited, and may be,for example, polystyrene, polypropylene, polyethylene, acrylic resins,glass, or metal. The vessel surface to be coated with the coating layerhas preferably been subjected to surface treatment, such as coronatreatment.

The coating layer may be formed at desired portions of the vesselsurface using at least one of the homopolymer of monomer (M) and thecopolymer of monomer (M) and another monomer, by, for example,dissolving the polymer in one of water, ethanol, methanol, isopropanol,and the like, or in a mixed solvent of water and ethanol, ethanol andisopropanol, or the like, and then soaking the vessel in the polymersolution, or spraying the polymer solution over the vessel.

When the copolymer has a functional group capable of chemical bonding,such as an epoxy, isocyanate, succinimide, amino, carboxyl, or hydroxylgroup, in order for such a functional group to be chemically reactedwith the amino, carboxyl, or hydroxyl group on the vessel surface, thevessel for embryoid formation may be prepared by dissolving a solutioncontaining the copolymer in a solvent that is not reactive with thefunctional group capable of chemical bonding, to chemically bond thecopolymer to the vessel surface to form the coating layer, and thenwashing away the unreacted polymer.

The method for forming embryoid bodies according to the presentinvention includes the steps of: (A) providing a vessel for embryoidformation having a coating layer formed from a compound having a PC-likegroup represented by the formula (1), on a vessel surface defining aregion for floating culture of ES cells, and (B) floating culturing EScells in the vessel for embryoid formation to form embryoid bodies.

The vessel provided in step (A) may be a vessel for embryoid formationaccording to the present invention, and all the vessels exemplifiedabove may be employed as the vessel provided in step (A).

The floating culture of ES cells in step (B) may be carried out byfloating culturing undifferentiated ES cells that have been cultured onfeeder cells, in the vessel for embryoid formation by a conventionalmethod under conventional conditions. Here, the culture liquid in thevessel for embryoid formation may be kept under static conditions orgently shaken.

The medium constituting the culture liquid may be a medium containingvarious growth factors used for the conventional hanging drop method,such as Iscove's modified Dulbecco's medium (IMDM medium).

The concentration of ES cells in the culture liquid may vary dependingon the size, shape, or the like, of the vessel for embryoid formationprovided in step (A), but may usually be in the range of 1.0×10² to1.0×10⁶ cells/mL. Specifically, when a 96-well plate is used as thevessel for embryoid formation, a preferred concentration of ES cells is1.0×10³ to 1.0×10⁵ cells/mL for formation of embryoid bodies with goodreproducivity.

EXAMPLES

The present invention will now be explained in more detail withreference to Examples and Comparative Examples, which do not intend tolimit the present invention. In the Examples and Comparative Examples,the P/C value on the vessel surface was determined in accordance withthe following method.

<Measurement of P/C Value on the Surface of Vessel for EmbryoidFormation>

Spectrum of each element was measured with an X-ray photoelectronspectroscope (trade name “ESCA-3300”, manufactured by SHIMADZUCORPORATION) at an X-ray irradiation angle of 90°, and from the obtainedpeak areas of phosphorus and carbon elements, the P/C value wascalculated in accordance with the following formula: P/C=Ap (peak areaof phosphorus element)/Ac (peak area of carbon element)

Synthesis Example 1

35.7 g of MPC and 4.3 g of n-butylmethacrylate (BMA) (MPC/BMA=80/20 (bymolar ratio)) were dissolved in 160 g of ethanol, placed in a four-neckflask, and bubbled with nitrogen for 30 minutes. 0.82 g ofazobisisobutyronitrile was added at 60° C., and reacted forpolymerization for 8 hours. The obtained polymer liquid was addeddropwise into 3 L of diethyl ether under stirring, and the resultingprecipitate was recovered by filtration, and vacuum dried at roomtemperature for 48 hours, to obtain 29.6 g of powder. The weight averagemolecular weight of the obtained powder measured by GPC under thefollowing conditions, was found to be 153000. Compositional analysis by¹H-NMR revealed that MPC/BMA=80/20 (by molar ratio). The powder isdesignated as copolymer (A).

<Conditions of GPC>

(1) Sample: A sample was dissolved in a chloroform/methanol (6/4 (byvolume)) mixed solvent containing 0.5 wt % lithium bromide to prepare a0.5 wt % polymer solution. The amount of the sample solution used was 20L.(2) Column: Two PLgel 5 μm MIXEDC-C columns arranged in series(manufactured by POLYMER LABORATORIES LTD.) were used at a columntemperature of 40° C., and a molecular weight calculating program withintegrator (GPC program for SC-8020) manufactured by TOSOH CORPORATIONwas used.(3) Eluting solvent: A chloroform/methanol (6/4 (vol %)) mixed solventcontaining 0.5 wt % lithium bromide was used, at a flow rate of 1.0mL/min.(4) Detection: Differential refractive index detector(5) Reference material: Polymethylmethacrylate (PMMA) (manufactured byPOLYMER LABORATORIES LTD.)

Synthesis Example 2

38.0 g of MPC and 2.0 g of glycidyl methacrylate (GMA) (MPC/GMA 90/10(by molar ratio)) were dissolved in 358 g of isopropanol, placed in afour-neck flask, and bubbled with nitrogen for 30 minutes. 2.18 g of atoluene solution of 20 wt % t-butyl peroxypivalate was added at 60° C.,and reacted for polymerization for 5 hours. The obtained polymer liquidwas added dropwise into 3 L of diethyl ether under stirring, and theresulting precipitate was recovered by filtration, and vacuum dried atroom temperature for 4.8 hours, to obtain 28.4 g of powder.Compositional analysis of the powder by ¹H-NMR revealed thatMPC/GMA=90/10 (by molar ratio). The weight average molecular weightmeasured by GPC under the same conditions as in Synthesis Example 1 wasfound to be 53000. The powder is designated as copolymer (B).

Synthesis Example 3

12.6 g of MPC, 8.6 g of BMA, and 6.0 g of GMA (MPC/BMA/GMA=30/40/30 (bymolar ratio)) were dissolved in 358 g of isopropanol, placed in afour-neck flask, and bubbled with nitrogen for 30 minutes. 2.18 g of atoluene solution of 20 wt % t-butyl peroxypivalate was added at 60° C.,and reacted for polymerization for 5 hours. The obtained polymer liquidwas added dropwise into 3 L of diethyl ether under stirring, and theresulting precipitate was recovered by filtration, and vacuum dried atroom temperature for 48 hours, to obtain 28.4 g of powder. Compositionalanalysis of the powder by ¹H-NMR revealed that MPC/BMA/GMA=30/40/30 (bymolar ratio). The weight average molecular weight measured by GPC underthe same conditions as in Synthesis Example 1 was found to be 42000. Thepowder is designated as copolymer (C).

Example 1-1

0.5 g of copolymer (A) synthesized in Synthesis Example 1 was dissolvedin 100 mL of ethanol to prepare a copolymer solution. 0.3 mL of thecopolymer solution was introduced into each well of a U-bottom 96-wellplate made of polystyrene, and then aspirated away. The plate was driedunder reduced pressure at 50° C. for 5 hours to give vessel (A) forembryoid formation.

The P/C value on the well surface having a coating layer of copolymer(A) of the vessel (A) for embryoid formation was measured. The result isshown in Table 1.

Example 1-2

A U-bottom 96-well plate made of polystyrene was subjected to coronatreatment in the air at the irradiation energy of J/cm² to generatecarboxyl groups on the surface. 0.5 g of copolymer (B) synthesized inSynthesis Example 2 was dissolved in 100 mL of isopropanol to prepare acopolymer solution. 0.3 mL of the copolymer solution was introduced intoeach well of the corona treated, U-bottom 96-well plate, and thenaspirated away. The carboxyl groups on the plate surface were reactedwith the epoxy groups of the copolymer at 60° C. for 3 hours. 0.3 mL ofa 0.2 M sodium thiosulfate aqueous solution was introduced into eachwell, and reacted at 25° C. for 24 hours for ring-opening the unreactedepoxy. Each well was washed three times with distilled water, and driedunder reduced pressure at 50° C. for 5 hours to prepare vessel (B) forembryoid formation.

The P/C value on the well surface having a coating layer of copolymer(B) of the vessel (B) for embryoid formation was measured. The result isshown in Table 1.

Example 1-3

Vessel (C) for embryoid formation was prepared in the same way as inExample 1-2, except that copolymer (B) was replaced with copolymer (C)synthesized in Synthesis Example 3.

The P/C value on the well surface having a coating layer of copolymer(C) of vessel (C) for embryoid formation was measured. The result isshown in Table 1.

Comparative Example 1

The P/C value on the well surface of an untreated, U-bottom 96-wellplate made of polystyrene was measured. The result is shown in Table 1.

TABLE 1 Vessel P/C ratio Example 1-1 Vessel (A) for embryoid 0.038formation Example 1-2 Vessel (B) for embryoid 0.074 formation Example1-3 Vessel (C) for forming 0.038 embryoid bodes Comparative Untreatedplate 0.000 Example 1

Example 2-1

Each well of vessel (A) for embryoid formation prepared in Example 1-1was plated with 0.2 mL of a suspension of mouse ES cells containing2×10⁴ cells/mL prepared in accordance with the following process. Afterculture at 37° C. in 5% CO₂ for 5 days, the development of embryoidbodies was observed under a phase contrast microscope. The result isshown in Table 2. A photocopy of the phase contrast micrograph is shownin FIG. 1.

In Table 2, the development of embryoid bodies was evaluated andindicated as A when an embryoid body of sufficient size fordifferentiation was formed; B when an embryoid body was formed but notof a sufficient size; and C when no embryoid body was formed.

<Preparation of Suspension of Mouse ES Cells> (1) Culture of FeederCells

As feeder cells, SIM mouse fibroblasts (abbreviated as STO cellshereinbelow) were used. The STO cells were cultured in Dulbecco'smodified Eagle's medium (abbreviated as DMEM medium hereinbelow,manufactured by GIBCO) supplemented with 25 units/mL of penicillin, 25g/mL of streptomycin, and 10 vol % of immobilized fetal calf serum(FCS). The cultured STO cells were treated with a 10 g/mL mitomycin Csolution (manufactured by SIGMA) for 3 hours, and a cell suspension wasprepared. The STO cell suspension, containing 5×10⁵ cells, was plated ineach well of a 6-well multidish, and cultured at 37° C. in 5% CO₂ for 16hours to prepare feeder cells.

(2) Culture of Mouse ES Cells

As ES cells, 129V mouse ES cells were used. The medium for ES cells wasa DMEM medium supplemented with 15% Knock Out (trade mark) serumreplacement (KSR: manufactured by GIBCO), 1 mM sodium pyruvate(manufactured by GIBCO), 0.1 mM nonessential amino acids (manufacturedby GIBCO), 0.1 mM 2-mercaptoethanol (manufactured by SIGMA), 25 units/mLof penicillin, 25 g/mL of streptomycin, and 1000 units/mL of murineleukemia inhibitory factor (mLIF: manufactured by CHEMICON) (abbreviatedas ES medium hereinbelow). 2×10⁵ cells/well of the ES cells were platedon the feeder cells prepared in paragraph (1) above, and cultured at 37°C. in 5% CO₂ for 3 days.

The mouse ES cells cultured in paragraph (2) above were released by acommon procedure using 0.1% trypsin-EDTA, and suspended in an IMDMmedium (manufactured by GIBCO, without mLIF) supplemented with 15% FCS,0.1 mM 2-mercaptoethanol (manufactured by SIGMA), 25 units/mL ofpenicillin, and 25 g/mL of streptomycin, to prepare a suspension ofmouse ES cells at a concentration of 2×10⁴ cells/mL.

Examples 2-2 and 2-3

The experimental procedures of Example 2-1 were followed, except thatvessel (A) for embryoid formation was replaced with vessel (B) or (C)for embryoid formation prepared in Example 2-2 or 2-3, respectively. Theresults are shown in Table 2.

Comparative Example 2

The experimental procedures of Example 2-1 were followed, except thatvessel (A) for embryoid formation was replace with an untreated 96-wellpolystyrene plate. The result is shown in Table 2. Further, thedevelopment of embryoid bodies was observed under a phase contrastmicroscope. A photocopy of the phase contrast micrograph is shown inFIG. 2.

Comparative Example 2-2

130 μL of phosphate buffer and 200 μL of mineral oil were introduced inadvance in each well of a flat bottom 96-well plate made of polystyrene,and then 50 μL of the 2×10⁴ cells/mL suspension of mouse ES cellsprepared above was plated in each well. After culture at 37° C. in 5%CO₂ for 5 days, the resulting embryoid bodies were transferred to aU-bottom 96-well plate made of polystyrene. Then phase contrastmicroscopic observation was made in the same way as in Example 2-1. Theresult is shown Table 2. A photocopy of the phase contrast micrograph isshown in FIG. 3.

Comparative Example 2-3

The experimental procedures of Example 2-1 were followed, except thatvessel (A) for embryoid formation was replaced with SUMILON celltightspheroid (trade mark, 96-well plate, manufactured by SUMITOMO BAKELITECO., LTD.) The result is shown in Table 2.

TABLE 2 Formation of Vessel embryoid bodies Example 2-1 Vessel (A) forembryoid A formation Example 2-2 Vessel (B) for embryoid A formationExample 2-3 Vessel (C) for embryoid A formation Comparative Example 2-1Untreated plate C Comparative Example 2-2 Hanging drop method BComparative Example 2-3 Spheroid plate B

Examples 3-1 to 3-3

The embryoid bodies prepared in Examples 2-1 to 2-3 were pipetted with0.1 mL of the medium, and transferred to a gelatin-coated dish preparedby the following process. Half of the medium was changed every 3 days.After culture at 37° C. in 5% CO₂ for 7 days, phase contrast microscopicobservation was made. The results are shown in Table 3.

In Table 3, the differentiation into cardiomyocyte was evaluated andindicated as A when beating cardiomyocytes were observed; B when a fewbeating cardiomyocytes were observed; and C when the operation was notpossible.

<Preparation of Gelatin-Coated Dish>

A 0.1 wt % aqueous solution of gelatin previously sterilized byautoclaving at 121° C. for 20 minutes, was uniformly spread over a24-well culture multidish. The multidish was refrigerated, and thegelatin solution was aspirated with an aspirator immediately before use.1 mL of IMDM medium (manufactured by GIBCO, without mLIF) supplementedwith 15% FCS, 0.1 mM 2-mercaptoethanol (manufactured by SIGMA), 25units/mL of penicillin, and 25 g/mL of streptomycin, was added to eachwell.

Comparative Example 3-1

The cells adhered to the plate bottom in Comparative Example 2-1 weretried to be transferred to a gelatin-coated dish, but were notsuccessful.

Comparative Examples 3-2 and 3-2

The experimental procedures of Example 3-1 were followed, except thatthe embryoid bodies prepared in Comparative Examples 2-2 (ComparativeExample 3-2) and 2-3 (Comparative Example 3-3) were used. The resultsare shown in Table 3.

TABLE 3 Differentiation into Vessel cardiomyocytes Example 3-1 Vessel(A) for embryoid A formation Example 3-2 Vessel (B) for embryoid Aformation Example 3-3 Vessel (C) for embryoid A formation ComparativeExample 3-1 Untreated plate C Comparative Example 3-2 Hanging dropmethod B Comparative Example 3-3 Spheroid plate B

Table 1 shows that the P/C values in Examples 1-1 to 1-3 are in therange of 0.038 to 0.074. This indicates that vessels (A) to (C) forembryoid formation were coated with a coating layer of a polymer havinga PC-like group. Table 2 indicates that culture of mouse ES cells invessels (A) to (C) for embryoid formation results in good formation ofembryoid bodies. Table 3 indicates that the embryoid bodies formed frommouse ES cells in vessels (A) to (C) for embryoid formation haveexcellent ability to differentiate into cardiomyocytes.

Further, from FIG. 1, it is understood that use of the vessel forembryoid formation according to the present invention results information of embryoid bodies of sufficient size for differentiation.From FIG. 2, it is understood that use of the untreated polystyrenevessel results in no formation of embryoid bodies. From FIG. 3, it isunderstood that the embryoid bodies formed by the hanging drop method isnot of sufficient size.

1-3. (canceled)
 4. A method for forming embryoid bodies comprising thesteps of: (A) providing a vessel for embryoid formation having a coatinglayer formed from a compound having a phosphorylcholine-like grouprepresented by the formula (1), on a vessel surface defining a regionfor floating-culture of embryonic stem cells:

wherein R¹, R², and R³ are the same or different groups, and each standsfor a hydrogen atom, an alkyl or hydroxyalkyl group having 1 to 6 carbonatoms; and n is an integer of 1 to 4; and (B) floating-culturingembryonic stem cells in said vessel for embryoid formation to formembryoid bodies.
 5. The method of claim 4, wherein said compound havinga phosphorylcholine-like group comprises a homopolymer of monomer (M)represented by the formula (2) having a phosphorylcholine-like group ora copolymer of monomer (M) and another monomer:

wherein R¹, R², and R³ are the same or different groups, and each standsfor a hydrogen atom, an alkyl or hydroxyalkyl group having 1 to 6 carbonatoms, R⁴ stands for an alkyl group having 1 to 6 carbon atoms, R⁵stands for a hydrogen atom or a methyl group; and n is an integer of 1to
 4. 6. The method of claim 4, wherein a ratio (P/C) of the amount ofphosphorus element P to the amount of carbon element C as measured byX-ray photoelectron spectroscopy on the vessel surface having saidcoating layer formed thereon is 0.002 to 0.3.
 7. (canceled)
 8. Themethod of claim 5, wherein said monomer (M) is selected from the groupconsisting of2-((meth)acryloyloxy)ethyl-2′-(trimethylammonio)ethylphosphate,3-((meth)acryloyloxy)propyl-2′-(trimethylammonio)ethylphosphate,4-((meth)acryloyloxy)butyl-2′-(trimethylammonio) ethylphosphate,5-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethylphosphate,6-((meth)acryloyloxy)hexyl-2′-(trimethylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(triethylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tripropylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tributylammonio)ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(tricyclohexylammonio) ethylphosphate,2-((meth)acryloyloxy)ethyl-2′-(triphenylammonio)ethylphosphate,2-((meth)acryloyloxy)propyl-2′-(trimethylammonio)ethylphosphate,2-((meth)acryloyloxy) butyl-2′-(trimethylammonio)ethylphosphate,2-((meth)acryloyloxy)pentyl-2′-(trimethylammonio)ethylphosphate, or2-((meth)acryloyloxy)hexyl-2′-(trimethylammonio) ethylphosphate.
 9. Themethod of claim 5, wherein said another monomer is selected from thegroup consisting of methyl(meth)acrylate, ethyl(meth)acrylate,butyl(meth)acrylate, 2-ethylhexyl(meth)acrylate, lauryl(meth)acrylate,stearyl(meth)acrylate, cyclohexyl(meth)acrylate, benzyl(meth)acrylate,phenoxyethyl(meth)acrylate, polypropylene glycol(meth)acrylate, styrene,methylstyrene, chloromethylstyrene, methyl vinyl ether, butyl vinylether, vinyl acetate, vinyl propionate, 2-hydroxyethyl(meth)acrylate,2-hydroxybutyl(meth)acrylate, 4-hydroxybutyl(meth)acrylate, acrylicacid, methacrylic acid, styrenesulfonic acid,(meth)acryloyloxyphosphonic acid, 2-hydroxy-3-(meth)acryloyloxypropyltrimethyl ammonium chloride, (meth)acrylamide, aminoethylmethacrylate,dimethylaminoethyl(meth)acrylate, polyethylene glycol (meth)acrylate,glycidyl(meth)acrylate, and mixtures thereof.
 10. The method of claim 5,wherein the weight average molecular weight of said homopolymer and saidcopolymer is 5000 to 5000000.